RNA interference targeting BxNDUFA2 impairs mitochondrial function and triggers oxidative stress to control pine wood...

Pine wilt disease (PWD), caused by the pine wood nematode (Bursaphelenchus xylophilus, PWN), is a devastating forest disease with limited eco-friendly control strategies, making novel species-specific control methods an urgent research priority. This study targets the oxidative phosphorylation (OXPHOS) pathway and identifies BxNDUFA2 (a key subunit of mitochondrial respiratory complex I) as a novel RNA interference (RNAi) target for PWN control. We synthesized dsBxNDUFA2, the double-stranded RNA specifically targeting BxNDUFA2, via a prokaryotic expression system and tested its effects on PWN. dsBxNDUFA2 significantly inhibited PWN survival, reproduction, and locomotor activity in a concentration-dependent manner. After 144 h treatment, corrected PWN mortality ranged 66.7%-78.6% at 100-1000 ng/μL, with an LT₅₀ of 29.3 h at 500 ng/μL. Nematode population dropped from 1.10 × 10⁴ (control) to 1.99 × 10³ (1000 ng/μL treatment), an 82% reduction. Electron microscopy revealed severe ultrastructural damage: body surface wrinkling, muscle atrophy, mitochondrial swelling, and cell membrane lysis. Biochemical assays showed dsBxNDUFA2 induced excessive reactive oxygen species (ROS) (47.7-fold fluorescence intensity increase at 1000 ng/μL vs untreated control) and malondialdehyde (MDA) (7.6-fold increase at 1000 ng/μL), with significantly downregulated superoxide dismutase (SOD) and catalase (CAT) activities, indicating severe oxidative stress and lipid peroxidation. Mechanistically, silencing BxNDUFA2 disrupted mitochondrial complex I function, causing energy metabolism disorders and oxidative stress-mediated cell damage. These findings confirm BxNDUFA2 as a highly promising RNAi target, providing a solid basis for developing green, species-specific, sustainable PWD control strategies.